Candidate genes and pathways downstream of PAX8 involved in ovarian high-grade serous carcinoma

Abstract

Understanding the biology and molecular pathogenesis of ovarian epithelial cancer (EOC) is key to developing improved diagnostic and prognostic indicators and effective therapies. Although research has traditionally focused on the hypothesis that high-grade serous carcinoma (HGSC) arises from the ovarian surface epithelium (OSE), recent studies suggest that additional sites of origin exist and a substantial proportion of cases may arise from precursor lesions located in the Fallopian tubal epithelium (FTE). In FTE cells, the transcription factor PAX8 is a marker of the secretory cell lineage and its expression is retained in 96% of EOC. We have recently reported that PAX8 is involved in the tumorigenic phenotype of ovarian cancer cells. In this study, to uncover genes and pathways downstream of PAX8 involved in ovarian carcinoma we have determined the molecular profiles of ovarian cancer cells and in parallel of Fallopian tube epithelial cells by means of a silencing approach followed by an RNA-seq analysis. Interestingly, we highlighted the involvement of pathways like WNT signaling, epithelial-mesenchymal transition, p53 and apoptosis. We believe that our analysis has led to the identification of candidate genes and pathways regulated by PAX8 that could be additional targets for the therapy of ovarian carcinoma.

Keywords: PAX8; RNA-seq; fallopian tube secretory epithelial cells; ovarian cancer; transcriptional networks.

Figure 1. MsigDB analysis of the most relevant genes differentially expressed between FT194 and SKOV-3 cells

(A) MsigDB for genes upregulated ≥ 2 fold in SKOV-3 cells. (B) MsigDB for genes downregulated ≥ 2 fold in SKOV-3 cells.

Figure 2. The Venn diagram of genes modulated upon PAX8 knockdown in SKOV-3 and FT194 cells

(A) Overlap and differences of downregulated genes following PAX8 silencing between SKOV-3 and FT194 cells. (B) Overlap and differences of upregulated genes following PAX8 silencing between SKOV-3 and FT194 cells.

Figure 3. Validation of representative genes by qRT-PCR analysis

(A) Expression levels of 13 genes measured on total RNA prepared from SKOV-3 and FT194 cells. The values are means ± SD of three independent experiments in duplicate, normalized by the expression of IP08 and expressed as fold change with respect to FT194 cells. (B and C) Expression levels of some representative genes measured on total RNA prepared from FT194 and SKOV-3 cells transiently transfected with PAX8 siRNA or scramble siRNA 24 h (white bars), 48 h (black bars) and 72 h (grey bars) after transfection. The values are means ± SD of three independent experiments in duplicate, normalized by the expression of IP08 and expressed as fold change with respect to the cells transfected with the scramble siRNA, whose value was set at 1.0. p-value was calculated by t-test 0.001 ≤ p ≤ 0.1.

Figure 4. Direct binding of PAX8 on the regulatory regions of putative target genes

Chromatin immunoprecipitation assays were performed to determine the binding of PAX8 to the 5′-flanking region of representative genes. Chromatin was subjected to quantitative real-time PCR analysis using appropriate primers (see Materials and Methods). Error bars indicate s.d. between two experiments performed in duplicate (p < 0,001).

Figure 5. Biological processes and pathways altered in siPAX8 cells

Gene ontology (GO) and Panther pathway anlysis have been performed using GeneCodis (http://genecodis.dacya.ucm.es/). (A) GO categories enriched for genes modulated upon PAX8 silencing in SKOV-3 cells. (B) GO categories enriched for genes modulated upon PAX8 silencing in FT194 cells. (C) Panther pathways enriched for genes modulated upon PAX8 silencing in SKOV-3 cells. (D) Panther pathways enriched for genes modulated upon PAX8 silencing in FT194 cells.

Figure 6. Pathway analysis of differentially expressed genes after PAX8 silencing

MsigDB software was used to identify the pathways most affected by the gene dysregulation. (A) MsigDB for SKOV-3 downregulated (black bars) and upregulated genes (gray bars). (B) MsigDB for FT194 downregulated (black bars) and upregulated genes (gray bars).